LB9
A comparison of the sensitivities of High Resolution Melting (HRM) analysis and pyrosequencing for mutation detection
Salih Ibrahem2, Rashmi Seth1, Fadhil Wakkas2, Brendon O'Sullivan3, Phillipe Taniere3, Mohammad Ilyas2
1Nottingham University Hospitals, NHS Trust, UK; 2Nottingham University, UK; 3University Hospitals, Birmingham NHS Foundation Trust, UK
Background and Aim
Mutation detection is important in the management of cancer. Several
platforms exist for mutation detection but there is no consensus on the best
method. High Resolution Melting (HRM) analysis and pyrosequencing are the
most versatile current methodologies and thus we undertook a comparative
analysis of the sensitivity of these techniques.
Method
DNA from HCT116 (a cell line mutant for KRAS and wild type for BRAF)
was mixed with DNA from Vaco5 (a cell line mutant for BRAF and wild type for KRAS)
to produce mixtures containing mutant alleles for each gene at a frequency of
25% / 12.5% / 6.25% / 3.125% / 1.625% / 0%. Each sample underwent
analysis for KRAS and BRAF by HRM and
pyrosequencing. Two colorectal cancers (CRC) showing KRAS
mutation by HRM but wild type by direct sequencing, underwent
pyrosequencing. Two CRCs showing PIK3CA mutation by HRM but wild
type by direct sequencing, underwent enrichment of mutant alleles by cold PCR
followed by direct sequencing.
Results
HRM analysis of KRAS showed that 3.125% mutant alleles could be
reliably detected whilst pyrosequencing could reliably detect 6.25% mutant
alleles. Analysis of BRAF by HRM showed that 3.125% mutant alleles
could be reliably detected whilst pyrosequencing could reliably detect 12.5%
mutant BRAF alleles. The cases of CRC previously considered as
false positives for KRAS mutation by HRM were shown by pyrosequencing
to be true positives. The cases of CRC previously considered as false
positives for PIK3CA mutation by HRM were shown to be true positives by
direct sequencing following cold PCR.
Conclusion
HRM is marginally more sensitive than pyrosequencing for detecting mutant
alleles and both are more sensitive that direct sequencing which can no longer
be regarded as gold standard. Cold PCR may enhance the
sensitivity of direct sequencing but it is a more cumbersome technique.
